Seven active males were recruited to examine changes in the serum concentration of S100, a proposed peripheral marker of BBB permeability, following prolonged exercise in temperate (T) and warm (W) conditions. Subjects were seated immersed to the neck in water at 35.0 ± 0.1°C (T) or 39.0 ± 0.1°C (W) for 30 min. Subjects then entered a room maintained at either 18.3 ± 1.8°C(T) or 35.0 ± 0.3°C (W) and completed 60 min of cycle exercise at 60 % VO₂ peak. Serum S100 concentration was elevated after exercise in the warm trial (+0.12 ± 0.10µg.L^-1; P = 0.02), but not after the temperate trial (P = 0.238). Water immersion and exercise elevated core temperature by 2.1 ± 0.5°C to 39.5 ± 0.3°C at the end of exercise in the W trial compared to a 0.9 ± 0.2°C increase during the T trial (P < 0.001). Weighted mean skin temperature was higher throughout the warm trial compared to the temperate trial (P < 0.001). Heart rate (P < 0.001), as well as blood glucose (P < 0.001) and lactate (P < 0.001) concentrations were elevated to a greater extent during exercise in the warm environment than in the temperate. Ratings of perceived exertion (P < 0.001) and thermal comfort (P < 0.001) were markedly higher throughout the W trial than in the T trial. The results of this study demonstrate that serum S100 was elevated following water immersion and prolonged exercise in a warm environment, suggesting that BBB permeability may be altered.